LIBRARY 

OF  THE 

UNIVERSITY  OF  CALIFORNIA. 

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tUOLOGY 

LIBRARY 

G 


DX3 

u  IS  ir>n 


The  University  of  Chicago 

K01M)KI>    MY  JOHN    1).   1!( )(  K  Kl  I'.LLEK 


VARIATION  AND  CARBOHYDRATE 

METABOLISM  OF  BACILLI  OF  THE 

PROTEUS  GROUP. 


A  DISSERTATION 

SUBMITTED  TO  THE  FACULTY 

OF  THE 
OGDEN  GRADUATE  SCHOOL  OF  SCIENCE 

IN  CANDIDACY  FOR  THE  DEGREE  OF 
DOCTOR  OF  PHILOSOPHY 

(DEPARTMENT  OF  BACTERIOLOGY) 


BY 


THOMAS  HAIGH  GLENN 


CHICAGO 
1911 


The  University  of  Chicago 

FOUNDED  BY  JOHN  D.  EOCKEFELLER 


VARIATION  AND  CARBOHYDRATE 


METABOLISM  OF  BACILLI  OF  THE 


PROTEUS  GROUP. 


A  DISSERTATION 

SUBMITTED  TO  THE  FACULTY 

OF  THE 
OGDEN  GRADUATE  SCHOOL  OF  SCIENCE 


(DEPARTMENT  OF  BACTERIOLOGY) 


BY 

THOMAS  HAIGH  GLENN 

\\ 


CHICAGO 
1911 


Introduction. 

It  has  been  shown  by  many  observers  that  changes  in  the  chemical 
and  physical  environment  of  micro-organisms  lead  to  marked  variations, 
not  only  in  the  morphology  of  the  cell,  but  also  in  its  physiological 
activities.  The  variations  in  the  form  and  staining  characteristics  of 
B.  diphtheria  e,  the  formation  of  long  and  short  chains  in  the  Strepto- 
cocci, the  appearance  of  capsules,  the  production  of  spores  and  devel- 
opment of  involution  forms  in  old  cultures  indicate  the  readiness  with 
which  unicellular  organisms  respond  to  their  external  environment. 

The  facility  with  which  micro-organisms  change  their  form  and  the 
mechanical  difficulties  involved  in  the  observation  of  variations  in  their 
morphology,  renders  any  classification  of  bacteria  based  on  morphology 
alone  very  unsatisfactory.  Recently,  the  bio-chemical  characteristics  of 
bacteria  have  played  an  important  part  in  the  study  of  bacterial  species. 
The  physiological  activities  of  bacteria  in  different  cultural  media  are 
easily  studied,  and  under  a  uniform  set  of  conditions  many  of  the 
characteristics  of  species  exhibit  a  remarkable  degree  of  constancy.  If 
uniform  conditions  are  not  adhered  to,  however,  variations  may  be 
obtained. 

Pere"  (1)  in  his  researches  on  lactic  acid  produced  by  micro- 
organisms found  that  the  same  organism  may  produce  lactic  acid  of  the 
opposite  optical  activity,  according  to  the  quantity  and  the  quality  of  the 
protein  used.  The  correctness  of  this  observation  has,  however,  been 
questioned  by  later  workers.  Kruse  (2)  found  that  Staphylococci 
lose  their  liquefying  power  after  prolonged  cultivation  under  anaerobic 
conditions.  Andrewes  and  H o r d e r  (3)  record  a  case  where  Strepto- 
cocci refused  to  attack  lactose  under  ordinary  conditions,  but  did  so 
readily  under  anaerobic  conditions.  Conn  (4)  starting  with  a  pure 
culture  of  a  micrococcus  was  -able  to  obtain  by  simply  replating  many 
times  and  selecting  from  the  number  of  colonies  on  the  plate,  the  one 
which  liquefied  most  rapidly  and  the  one  which  liquefied  most  slowly, 
a  rapidly  liquefying  culture  and  one  which  hardly  liquefied  at  all. 
Smith  (5)  records  similar  results  with  Proteus  vulgaris.  Peckham 
(6)  has  shown  that  organisms  that  may  not  have  the  power  to  produce 
indol  may  develop  this  power  if  allowed  to  grow  in  suitable  media. 
T  w  o  r  t  (7),  by  growing  organisms  in  a  fluid  medium  containing  a  sugar 
that  they  had  not  been  previously  able  to  ferment,  was  able  to  cause 
these  organisms  to  ferment  the  sugar.  Goodman  (8)  states  that  he 
was  able  by  a  gradual  process  of  selection  of  impressed  variations, 

236712  * 


to  modify  greatly  the  acid  producing  characteristics  of  the  diphtheria 
group  of  organisms. 

The  present  paper  is  a  report  of  the  results  of  an  investigation  upon 
the  variation  and  carbohydrate  metabolism  of  the  bacilli  of  the  P  r  o  t  e  u  s 
group.  In  view  of  the  fact  that  this  group  of  organisms  is  found  widely 
distributed  under  varied  conditions  in  nature,  it  offers  a  promising  field 
for  investigation. 

Historical. 

The  members  of  this  group  of  micro-organisms  were  first  isolated 
by  Hauser  (9)  who  regarded  them  as  the  chief  bacteria  concerned  in 
the  process  of  putrefaction.  He  isolated  three  forms  which  he  originally 
regarded  as  distinct  species,  but  his  later  observations  led  him  to  believe 
that  all  three  forms  are  but  varieties  of  the  same  species.  Since  then, 
they  have  been  isolated  many  times  from  sewage  by  many  different  in- 
vestigators. Schnitzer  (10)  isolated  a  nember  of  the  Proteus  group 
from  a  case  of  cystitis.  Flexner  (11)  found  it  in  a  case  of  peritonitis, 
and  Reed  (12)  found  aProteus  organism  associated  with  the  P  n  e  u  m  o  - 
coccus  in  a  case  of  croupous  pneumonia.  Levy  (13)  isolated  aProteus 
form  from  a  sample  of  beer  yeast.  Wyss  (14)  obtained  B.  vulgaris 
from  dead  fish.  Silberschmidt  (15),  Gluckman  (16),  and  others 
have  isolated  members  of  the  group  from  infected  meat. 

Ward  (18)  studied  a  number  of  organisms  of  this  group  which  he 
isolated  from  the  Thames  and  came  to  the  conclusion  that  Hauser's 
three  species  of  Proteus,  together  with  several  others  isolated  by 
himself,  are  merely  variations  of  one  species.  He  considers  them  similar 
in  size  and  form,  mode  of  growth,  formation  of  zooglea,  etc.,  but  as 
varying  in  detail  as  to  rapidity  of  growth  and  liquefaction,  and  consequent 
differences  in  the  extent  and  appearance  of  the  colony,  and  as  to  the 
intensity  of  pigmentation.  Fuller  and  Johnson  (19)  regard  all  non- 
fluorescent,  non-chromogenic  gelatin  liquefying  bacteria  forming  proteus- 
like  colonies  on  gelatin,  as  belonging  to  the  Proteus  group.  Ford 
(20)  includes  under  the  Proteus  group  all  organisms  which  liquefy 
blood  serum,  casein,  and  gelatin,  produce  cloudiness  in  broth  but  no 
scum,  render  milk  acid,  then  alkaline,  and  which  ferment  dextrose  with 
gas  formation.  J  o  r  d  a  n  (21)  includes  under  the  P  r  o  t  e  u  s  group,  organisms 
which  ferment  sucrose  and  dextrose,  rarely  lactose,  which  are  for  the 
most  part  vigorously  proteolytic,  rapidly  liquefying  gelatin  and  blood 
serum  and  precipitating  and  then  dissolving  casein. 

All  of  the  above  authors  seem  to  lay  special  stress  on  the  proteolytic 
and  fermentative  properties  of  this  group.  It  becomes  essential,  therefore, 
to  endeavour  to  establish  the  factors  which  may  influence  either  one  of 
these  characteristics. 

Source  of  cultures. 

The  organisms  used  in  this  investigation  were  obtained  from  the 
laboratories  of  the  University  of  Michigan,  University  of  Pennsylvania, 
University  of  Illinois,  University  of  Chicago,  the  laboratory  of  the  Public 
Health  and  Marine  Hospital  Service,  Washington  and  from  Krai's 
laboratory. 

All  cultures  were  rejuvenated  according  to  the  method  of  Fuller 
and  Johnson.  Transfers  were  made  from  the  gelatin  plates  to  slant 
agar.  These  were  incubated  for  24  hours  and  used  to  inoculate  all  the 


ordinary  media.  The  cultural  characteristics  of  each  organism  are  given 
in  table  No.  1. 

It  will  be  seen  from  an  examination  of  the  table  that  acid  is  produced 
readily  by  some  of  the  cultures,  while  others  produce  very  little  or  none 
at  all.  If  the  acid  producing  cultures  and  the  non-acid  producing 
organisms  of  this  group  belong  to  the  same  species,  it  was  thought 
possible  to  produce  by  selection  from  the  descendants  of  the  acid  pro- 
ducing variety  an  organism  which  would  produce  acid  readily  and  one 
which  would  not  produce  acid  at  all.  Cultures  1  and  2  were  selected. 
These  were  plated  out  in  gelatin  and  typical  colonies  were  fished  out 
and  transferred  to  slant  agar  tubes.  These  tubes  were  incubated  for 
24  hours  and  at  the  end  of  that  time  a  sub-culture  in  broth  was  made, 
and  from  each  of  the  24  hour  broth  cultures  a  single  bacillus  was  ob- 
tained by  Barber's  method  (22).  This  was  done  in  order  to  make 
sure  that  all  of  the  organisms  would  be  descendants  of  a  single  cell, 
so  that  the  individual  inheritance  might  be  studied  instead  of  averages. 

Goodman,  in  his  experiments  with  diphtheria  bacilli  made  transfers 
direct  from  sugar  broth  to  sugar  broth.  The  direct  influence  of  environ- 
ment, as  has  been  pointed  out  by  Win  slow  and  Walker  (23),  was 
not  entirely  excluded.  These  authors  attempted  to  exclude  this  factor 
in  their  study  of  the  variations  in  the  paratyphoid  bacillus  by  plating 
out  each  culture  on  a  series  of  gelatin  plates  and  inoculating  100  agar 
tubes  from  100  separate  colonies  of  each  strain.  Dextrose  broth  tubes 
were  then  inoculated  from  the  agar  slants  and  the  acidity  in  each  of 
the  sugar  broth  tubes  was  determined  by  titration  after  72  hours. 
Instead  of  making  further  inoculations  from  the  broth  tubes,  those  agar 
cultures  were  selected  which  in  broth  had  shown  the  highest  acidity  for 
their  respective  types.  These  were  plated  out  and  from  the  colonies  on 
the  plates  new  agar  streaks  were  made. 

Win  slow  and  Walker  (23),  while  excluding  the  factor  of  environ- 
ment, carried  their  cultures  through  three  generations  only  and  thus  did 
not  get  the  benefit  of  the  accumulation  of  differences  which  might  have 
taken  place  if  the  number  of  transfers  had  been  increased. 

In  the  present  investigation,  the  cultures  derived  from  a  single  cell 
were  plated  out  and  three  series  of  cultures  were  made.  In  the  first 
series,  twenty  separate  colonies  were  isolated  and  inoculated  into  twenty 
sugar-free  broth  tubes  to  which  1%  of  dextrose  had  been  added.  After 
three  days  incubation  at  37°,  the  acidity  was  determined  by  titration 

with  :r~:NaOH,  using  phenolphthalein  as  an  indicator.    The  tube  giving 

the  maximum  acidity  and  that  showing  the  minimum  acidity  were  plated 
out  and  the  dextrose  broth  tubes  were  inoculated  from  each  of  the 
plates,  a  separate  colony  being  fished  out  for  each  tube. 

In  the  second  series,  the  cultures  were  plated  out  and  10  agar  tubes 
were  inoculated  from  ten  separate  colonies.  These  tubes  where  incubated 
for  24  hours  at  37°  C.  and  a  tube  of  1%  dextrose  broth  was  inoculated 
from  each.  The  dextrose  tubes  were  incubated  at  37  °  C.  for  three  days 
and  titrated  as  in  the  first  series.  The  tubes  giving  the  maximum  and 
minimum  of  acid  were  selected.  Wishing  to  avoid  all  chances  of  dealing 
with  organisms  that  might  have  acquired  some  tolerance  to  degrees  of 
acidity  produced  in  the  medium  by  their  own  growth,  all  such  were 
discarded;  but  the  two  agar  streaks  derived  from  the  races  that  had 
shown  the  highest  and  the  lowest  acidity  for  their  respective  types,  were 


4     — 


used.  These  streaks  were  plated  out  and  from  the  colonies  on  the 
plates  new  agar  streaks  were  made  and  dextrose  broth  tubes  inoculated 
from  these  as  before.  In  no  case  either  in  the  plate  or  on  the  agar 
slant  was  the  culture  allowed  to  come  in  contact  with  sugar  until  in- 
oculated into  broth. 

In  the  third  series  of  cultures,  the  cultures  were  plated  out  as  in 
the  other  series,  but  the  colonies  were  transferred  direct  to  broth  tubes 
containing  1%  of  dextrose.  The  acidity  was  determined  as  before,  but 
in  this  series  the  culture  in  the  tubes  producing  the  highest  and  the 
lowest  amount  of  acidity  were  inoculated  direct  into  sugar  broth  without 
plating  out,  except  to  determine  the  purity  of  the  cultures. 

In  the  tables  are  given  the  averages  of  the  acid  produced  in  the 
high  and  low  series.  In  most  cases,  the  organism  selected  in  the  high 
series  produced  a  higher  percentage  of  acid  than  the  average,  and  the 
organism  selected  in  the  low  series  produced  a  lower  percentage  of 
acidity  than  is  shown  by  the  average  of  the  series,  but  any  true  variation 
should  be  transferred  to  all  of  the  descendants,  so  that  the  average  per- 
centage of  acid  produced  represents  a  fairly  good  index  of  the  variation 
of  the  acid  producing  power  of  the  organism.  From  these  tables,  it  is 

Proteus  vulgaris  1. 


No. 

Series  I 

Series  II 

Series  III 

High 

Low 

High 

Low 

High 

Low 

1 

2.29 

2.29 

2.29 

2.29 

2.35 

2.35 

2 

2.30 

2.28 

2.25 

2.22 

2.37 

2.29 

3 

2.34 

2.31 

2.40 

2.11 

2.19 

2.19 

4 

2.35 

2.29 

2.29 

2.30 

2.28 

2.17 

5 

2.4 

2.35 

2.30 

2.28 

2.34 

2.30 

6 

2.33 

2.1 

2.35 

2.25 

2.14 

2.05 

7 

2.2 

2.05 

2.31 

2.17 

1.98 

1.93 

8 

1.9 

1.85 

1.96 

1.93 

2.08 

1.98 

9 

2.17 

2.05 

1.96 

1.96 

2.05 

2.05 

10 

1.91 

1.95 

1.98 

1.94 

2.07 

2.08 

11 

1.95 

1.92 

2.22 

2.20 

2.14 

2.11 

12 

1.93 

1.9 

1.93 

1.94 

2.03 

1:98 

13 

2.03 

2.03 

2.00 

2.04 

1.91 

1.90 

14 

2.15 

2.03 

1.90 

1.89 

1.90 

1.89 

15 

2.13 

2.07 

2.16 

2.07 

1.88 

1.86 

16 

209 

2.09 

2.14 

2.12 

1.80 

1.78 

17 

2.05 

2.07 

2.08 

2.01 

1.80 

1.78 

18 

1.91 

1.86 

2.02 

2.00 

1.63 

1.64 

19 

1.97 

188 

1.72 

1.64 

1.65 

1.65 

20 

1.94 

1.81 

1.73 

1.66 

2.00 

1.99 

21 

1.94 

1.85 

1.58 

1.48 

1.82 

1.72 

22 

1.75 

1.73 

1.60 

1.59 

1.73 

1.71 

23 

1.74 

1.71 

1.74 

1.65 

1.94 

1.85 

24 

1.72 

1.68 

1.82 

1.88 

2.02 

1.96 

25 

1.55 

1.49 

2.04 

1.97 

1.97 

1.96 

26 

1.58 

1.56 

1.69 

1.67 

1.99 

1.98 

27 

1.61 

1.53 

1.70 

1.61 

2.03 

2.01 

28 

1.66 

1.61 

1.71 

1.80 

29 

1.62 

1.63 

30 

1.72 

1.68 

31 

2.03 

1.92 

32 

1.76 

1.71 

33 

2.07 

2.03 

34 

1.69 

1.68 

35 

1.75 

1.75 

Proteus  vulgaris  II. 


No. 

Series  I 

Series  II 

High             Low 

High 

Low 

1 

2.3 

2.3 

2.4 

2.4 

2 

2.2 

2.18 

2.4 

2.37 

3 

2.3 

2.3 

2.20 

2.16 

4 

2.38 

2.28 

2.08 

2.12 

5 

2.38 

2.41 

2.02 

1.94 

6 

1.97 

1.91 

1.87 

1.78 

7 

2.12 

2.14 

2.04 

1.96 

8 

1.84 

1.78 

1.90 

1.90 

9 

1.89 

1.83 

1.99 

2.01 

10 

1.9 

1.86 

2.1 

1.84 

11 

2.04 

1.99 

2.00 

1.84 

12 

1.89 

1.86 

1.91 

1.88 

13 

1.93 

1.92 

2.15 

2.10 

14 

1.88 

1.88 

1.78 

1.75 

15 

1.95 

1.91 

1.87 

1.86 

16 

2.02 

1.93 

1.79 

1.77 

17 

1.92 

1.89 

1.66 

1.55 

18 

1.63 

1.65 

1.68 

1.68 

19 

1.7 

1.69 

1.83 

1.81 

20 

1.68 

1.69 

1.95 

2.09 

21 

1.51 

1.5 

1.72 

1.68 

22 

1.52 

1.56 

1.97 

1.94 

23 

1.60 

1.64 

2.04 

2.00 

24 

1.77 

1.81 

1.96 

1.80 

25 

1.56 

1.57 

2.02 

1.99 

26 

1.57 

1.59 

1.91 

1.90 

27 

1.68 

1.56 

1.92 

1.91 

28 

1.82 

1.8 

1.92 

1.90 

29 

1.98 

1.83 

30 

1.92 

1.91 

31 

1.86 

1.83 

32 

1.91 

1.88 

evident  that  very  little,  if  any,  modification  of  either  of  the  organisms  has 
taken  place  as  a  result  of  the  selection  of  slight  variations  in  acid 
production.  In  all  cases,  there  is  a  slight  decrease  in  the  power  to 
produce  acid,  but  this  may  be  explained  by  the  fact  that  the  organism 
in  some  cases  was  in  contact  with  acid  so  long  that  all  of  its  powers 
may  have  been  inhibited.  In  the  cases  where  they  did  not  come  in  con- 
tact with  acid  at  all,  the  power  to  ferment  sugar  was  gradually  decreasing 
as  a  result  of  growth  on  sugar-free  media.  The  latter  conclusion  seems 
•justifiable  on  account  of  the  fact  that  organisms  which  have  been  grown 
on  sugar-free  media  for  some  time  developed  a  higher  power  of  acid 
production  when  cultivated  for  a  short  time  on  media  containing  sugar, 
although  this  power  could  be  decreased  again  if  the  growth  on  sugar 
media  was  prolonged,  due  to  coming  in  contact  with  acids  produced  by 
the  fermentation  of  the  sugar. 

Since,  selection  of  slightly  impressed  variation  in  organisms  Nos.  1 
and  2  failed  to  produce  a  non-acid  producing  culture,  the  cultures  which 
orginally  produced  no  acid  were  grown  on  media  containing  2%  of 
dextrose  for  varying  lengths  of  time.  I  succeeded  in  getting  two  cultures 
to  produce  acid,  but  all  the  other  cultures  labeled  Zenkeri  and  mira- 
bilis  failed  to  produce  any  degree  of  acidity  after  prolonged  cultivation 
on  sugar  media.  It  is  possible  that  continued  transferring  of  these 
organisms  from  sugar  broth  to  sugar  broth  would  cause  them  either  to 


-     6     — 
Table  I. 


Morphology 

Cultural 

£                D«7 

N  utrient 

Nutrient 

Gelatine 

I 

broth  tube 

agar  tube 

plate 

pfi 

s 

Name  of 
organism 

Source 

_§ 

1  .< 

_0 

* 

1 
Pq 

£  * 
|| 

"o 

I 

0 

a. 
OQ 

B 

3 

•3 

1 

s 

5 

a 

13 
J» 

_g 

*c 

II 

-4-3      ?- 

O   cS 

1  ** 

H 

^ 

,f~1       QT* 

5 

0 

1 

B.  vulgaris  I 

U.  of  Chicago 

+ 



+ 

_ 

+ 

+ 

2 

B.         „       II 

n     n             n 

-j- 

— 

-[- 

— 

— 

4. 

— 

— 

4_ 

3 

B.  mirabilis 

n     n             n 

4- 

— 

4- 

— 

— 

4_ 

— 

— 

4. 

4 

B.  Zenkeri 

n     n             n 

4- 

— 

_|- 

— 

— 

4_ 

— 

— 

4. 

5 

B.  cloacae  I 

n     n             n 

4- 

— 

-|. 

— 

— 

4_ 

— 

— 

— 

6 

B.        „       II 

n     n             n 

4- 

— 

-f- 

— 

— 

4. 

— 

— 

— 

7 

B.        „      III 

n     n             n 

4- 

— 

_(- 

— 



4. 



— 



8 

B.        „ 

Blood  of  Eng.  Sparrow 

4- 

— 

4- 

— 

— 

4_ 

— 

— 

— 

9 
10 

B.  vulgaris 
B.  mirabilis 

Hygienic  Lab. 

n                 n 

+ 

— 

+ 

— 

— 

1 

— 

— 

+ 

11 

B.  Zenkeri 

»                 n 

4- 

— 

-|. 

— 

— 

4. 

— 

— 

4. 

12 

B.  cloacae 

n                 n 

4_ 

— 

+ 

— 

— 

4. 

— 

— 

— 

13 

B.  vulgaris 

U.  of  Mich. 

4- 

— 

_|- 

— 

— 

4_ 

— 

— 

4. 

14 

B.        „ 

U.  of  Illinois 

4- 

— 

_|- 

— 

— 

4_ 

— 

— 

4. 

15 

B.  Zopfi 

n     n           " 

4- 

— 

-j- 

— 

— 

4. 

— 

— 

4_ 

16 

B.  vulgaris 

U.  of  Penn. 

4- 

— 

4- 

— 

— 

4. 

— 

— 

4. 

17 

B.  mirabilis 

n     n           n 

4- 

— 

-|- 

— 

— 

4- 

— 

— 

4_ 

18 

B.  Zenkeri 

n     n           » 

-f 

— 

4. 

— 

— 

4- 

— 

— 

4. 

19 

B.  cloacae 

n     n          n 

4- 

— 

4. 

— 

— 

4- 

— 

— 

— 

20 

B.  vulgaris 

Krai's  Lab. 

4- 

— 

4. 

— 

— 

4_ 

— 

— 

4_ 

21 

B.  mirabilis 

M 

_|_ 

— 

4. 





4. 





4. 

22 

B.  Zenkeri 

n            n 

4- 

— 

4_ 

— 



4_ 

— 

— 

4_ 

23 

B.  Zopfi 

n            n 

4- 

— 

4- 

— 

— 

4- 

— 

— 

4- 

24 

B.  cloacae 

51                 11 

+ 

— 

+ 

— 

— 

+ 

— 

— 

— 

regain  their  power  of  acid  production  or  develop  this  power.  A  series 
of  20  transfers  with  a  lapse  of  two  days  between  each  failed  to  cause 
them  to  ferment  sugar  to  any  appreciable  degree.  It  should  be  stated, 
however,  that  all  the  organisms  labeled  Zopfii,  Zenkeri,  and  mira- 
bilis, with  the  exception  of  Nos.  3  and  17,  grew  very  sparingly  on  all 
the  laboratory  media,  although  all  gave  the  characteristic  colonies  on 
gelatin  plates. 

All  the  organisms  which  fermented  dextrose  readily,  fermented  sac- 
charose, mannose,  and  galactose,  but  failed  to  ferment  lactose,  even  when 
grown  in  lactose  broth  for  several  days.  In  the  fermentation  tubes  con- 
taining lactose  broth  inoculated  with  these  organism,  there  appeared  good 
growth  in  the  open  arm,  but  none  whatever  in  the  closed  arm.  This 
suggested  the  idea  that  it  is  much  easier  for  these  organisms  to  get  their 
oxygen  from  the  air  than  from  lactose  and,  therefore,  this  sugar  is  not 
attacked.  If  this  conclusion  were  correct,  it  was  thought  possible  to 
cause  the  non-lactose  fermenting  organisms  to  ferment  this  sugar  if  they 
were  deprived  of  all  other  sources  of  oxygen. 

Flasks  each  containing  100  c.  c.  of  sugar  free  broth,  to  which  2% 
of  lactose  had  been  added,  were  inoculated  with  strains  of  Proteus 
vulgaris  which  had  failed  repeatedly  to  ferment  lactose  under  aerobic 


Table  I. 


Biology 


features 


Gelatine 
stab 


+ 


+ 


+ 


Potato 
tube 


+ 


+ 


Ferment. 
tube 


»•*-; 

5 


.a  s 


^o 

.g  S 

2 
o 


-W     Cj 

:|i 

&  s 

o_g 


S 


+ 


Biochemical  features 


o 


+ 


4- 


+ 


+ 


Lique- 
faction 


Gas 

production 


G 


O 


"8 


Milk 


ed 


-fc 


Nutrient 
agar  tubes 


ah 


omogenes 


conditions.  The  flasks  were  then  placed  in  jars  from  which  the  oxygen 
had  been  removed  by  pyrogallic  acid  and  NaOH.  The  jars  were  allowed 
to  stand  at  room  temperature  for  6  days,  when  the  flasks  were  removed 

N 

and  their  contents  titrated  against  ^  NaOH  to  determine  the  acidity.    In 

&\) 

every  case,  fermentation  had  taken  place  as  was  shown  by  the  difference 
between  the  acid  in  the  inoculated  tube  and  that  of  a  sterile  control 
which  was  placed  under  anaerobic  conditions  with  the  inoculated  flasks. 
The  amount  of  acid  produced  was  increased  by  inoculating  a  loopful  of 
broth  obtained  from  the  lactose  broth  flask  which  showed  slight  amount 
of  fermentation,  into  another  lactose  broth  flask  containing  100  c.  c.  of 
broth.  These  were  placed  under  anaerobic  conditions  as  in  the  previous 
case  and  tested  after  6  days.  The  experiment  was  carried  through  five 
generations  and  the  results  are  shown  in  the  following  table. 

Duplicate  flasks  grown  under  aerobic  conditions  at  the  same  tempe- 
rature, failed  to  develop  any  acid.  Fermentation  tubes  inoculated  with 
these  three  strains  of  Proteus  and  placed  under  anaerobic  conditions 
showed  a  growth  not  only  in  the  open  arm,  but  also  in  the  closed  one. 
This  would  seem  to  indicate  that  Proteus  vulgaris  can  obtain  its 
oxygen  from  the  air  much  more  easily  than  from  lactose,  but  when  it 


Table  No.  2. 


Per  cent. 

Per  cent. 

Per  cent. 

Per  cent. 

Per  cent. 

Organism 

of  acid  after 

of  acid  after 

of  acid  after 

of  acid  after 

of  acid  after 

1st  transfer 

2d  transfer 

3d  transfer 

4th  transfer 

5th  transfer 

Control 

0 

0 

0 

0 

0 

Proteus  vulgaris  I 

0.6 

1 

0.8 

1 

1.2 

„        H 

0.6 

1 

0.8 

0.8 

1.3 

»              »        1" 

0.6 

1 

1 

1.9 

5.1 

can  no  longer  obtain  oxygen  from  the  air  it  splits  up  lactose  with  acid 
formation. 

Effect  of  carbohydrates  on  protein  metabolism. 

Protein  digestion  may,  according  to  Peckh am  (6),  be  approximately 
estimated  in  many  members  of  the  colon  group  by  the  production  of 
indol.  Per 6  (1)  states  that  a  positive  reaction  for  indol  proves  the 
disappearance  of  sugar  in  the  media.  Grape  sugar,  Kruse  (2)  thinks, 
is  responsible  for  the  variation  in  indol  production,  although  he  does 
not  state  whether  the  variation  is  produced  by  the  sugar  itself  or  its 
decomposition  products.  Smith  (5)  thinks  the  absence  of  indol  in  sugar 
media  is  due  to  the  presence  of  acid.  Where  acid  is  present  no  indol 
is  produced,  but  where  acid  is  absent  indol  may  be  formed.  A  u  e  r  - 
bach  (24)  showed  that  the  acid  products  of  fermentation  set  up  by 
certain  bacteria  inhibit  the  formation  of  the  proteolytic  ferment. 
Kalischer  (25),  in  experiments  to  determine  whether  the  splitting  of 
casein  is  due  to  the  ferment  or  the  living  cell,  found  that  the  ferment 
was  able  to  produce  peptone,  leucin,  tyrosin,  as  well  as  ammonia  and 
oxyacids,  and  is  in  all  probability  a  tryptic  ferment.  Cacace  (26)  came 
to  the  conclusion  that  proteolysis  in  bacteria  is  similar  to  that  in  higher 
animals.  Wherry  (27),  working  with  the  cholera  spirillum,  found  that 
acids  produced  from  glucose,  maltose  and  saccharose  rapidly  killed  the 
cholera  spirillum,  while  those  from  lactose  and  starch  are  not  toxic. 
This  author  seems  to  think  that  the  proteolytic  ferment  of  the  cholera 
spirillum  is  a  tryptic  ferment.  Marshall  (28)  observed  that  Bacillus 
coli  grown  at  37°  C  for  five  days  in  100  c.  c.  of  peptone  beef  broth 
containing  20%  of  lactose  failed  to  produce  indol. 

From  experiments  already  cited,  it  is  evident  that  Proteus  vul- 
garis, under  aerobic  conditions,  does  not  produce  acid  in  lactose  media, 
while  acid  is  readily  produced  in  dextrose  broth.  If  Smith's  conclusion 
were  correct,  it  was  thought  that  dextrose  or  any  other  sugar  which  can 
be  fermented  by  Proteus  vulgaris,  should  inhibit  the  production 
of  indol,  while  lactose  or  any  other  carbohydrate  not  fermented  by  this 
organism  should  have  no  eifect  on  indol  production.  A  series  of  flasks 
were  prepared,  each  containing  about  100  c.  c.  of  distilled  water,  to  which 
the  percentage  of  peptone  and  carbohydrate  given  in  the  table  had  been 
added.  Each  flask  was  inoculated  with  one  loopful  of  a  homogeneous 
suspension  of  the  bacilli  and  placed  in  the  incubator  at  37  °.  Five  c.  c. 
of  broth  was  removed  after  48  hours  and  24  hours  thereafter  for  8  days, 
and  tested  for  indol  with  paradimethyl  benzaldehyde  solution  *). 


1)      4  parts  paradimethyl  benzaldehyde. 
80      „     hydrochloric  acid. 
360      „     alcohol,  95%- 


—     9     — 

Table  No.  3. 
Effect  of  carbohydrates   on  the  production  of  indol. 


-1-1 

o> 

„_  I 

o  a 

4 

ti 

Organism 

Per  cent. 
Peptone 

Per  cent. 
Dextrose 

-t>    10 
O    OQ 

Per  cent. 
Saccharos 

|| 

P^00 

53  'E 
o  g 

55  £? 

Indol  produced 

Hours 

48|72 

96 

120 

144 

168 

192 

216 

I 

i 

Prot.  vulg. 

0.5 

0 

0 

0 

0 

0 

4- 

4- 

4- 

4- 

4- 

4- 

4- 

4. 

2 

1 

0 

0 

0 

0 

0 

-j- 

4_ 

4_ 

4. 

4_ 

4. 

4. 

4. 

3 

2 

0 

0 

0 

0 

0 

4- 

4- 

4- 

4- 

4- 

4_ 

4. 

4_ 

4 

0.5 

0.5 

0 

0 

0 

0 

— 

— 

— 

— 

— 

— 

— 



5 

1 

0.5 

0 

0 

0 

0 

— 

— 

— 

— 

— 

— 

— 



6 

2 

0.5 

0 

0 

0 

0 

— 

— 

— 

— 

— 

— 

— 

— 

7 

1 

1 

0 

0 

0 

0 

— 

— 



— 

— 







8 

2 

1 

0 

0 

0 

0 

— 

— 

— 

— 

— 

— 

— 

— 

9 

2 

2 

0 

0 

0 

0 

— 

— 

— 

— 



— 

— 



10 

0.5 

0 

0.5 

0 

0 

0 

4- 

4. 

4- 

4. 

4. 

4. 

4. 

4. 

11 

0.5 

0 

2 

0 

0 

0 

4- 

-1- 

4_ 

4. 

4. 

4- 

4. 

4. 

12 

1 

0 

2 

0 

0 

0 

4- 

4- 

4. 

4- 

4_ 

4. 

4_ 

4. 

13 

2 

0 

2 

0 

0 

0 

4- 

4- 

-|_ 

4. 

4_ 

-4- 

4_ 

4. 

14 

1 

0 

0 

0.5 

0 

0 

— 

— 

— 

— 

-j. 

± 

-t- 

i1) 

15 

1 

0 

0 

1 

0 

0 

— 

— 

— 

— 

— 

— 

— 

16 

1 

0 

0 

2 

0 

0 

— 

— 

— 

— 

— 

— 

— 



17 

1 

0 

0 

0 

1 

0 

-l_ 

4. 

_l_ 

4. 

4. 

4_ 

4. 

4. 

18 

1 

0 

0 

0 

0 

0.5 

— 

4_ 

_l_ 

4. 

4. 

4_ 

4. 

4. 

19 

] 

0 

0 

0 

0 

1.0 

— 

4- 

4- 

4- 

4- 

4- 

4- 

4- 

The  table  shows  that  the  production  of  indol  takes  place  in  the 
presence  of  peptone  solution,  glycerin,  lactose,  and  starch ;  but  saccharose 
and  dextrose  inhibit  its  production.  In  a  second  series  of  experiments 
B.  coli  and  Prot.  vulgaris  were  used  for  inoculation.  The  solutions 
were  made  up  as  in  the  first  experiment  and  at  the  end  of  four  days 
all  the  flasks  were  titrated  against  n/10  NaOH.  The  results  are  given 
in  Table  No.  4. 

Table  No.  4. 
Effect  of  carbohydrates  on  the  production  of  indol. 


"a 

0  § 

d'S 

Or- 

-|J   O) 

c  a 

S3 

if 

0) 

*»  a> 

C   co 

cj  o 

-w  a 
a.-s 

80 

M-S 

J.s« 

"S  co   S 

-M     >* 

Q     S        . 

SO   co 
•**  >> 
,~  as 

Indol  produced 

gan 

£& 

So 

£  ^ 

0?^ 

o^S 

PH 

?<!Jk 

*f* 

OH  cs 

Hours 

w 

cc 

PH  ° 

24]48|72|96|120|144|168|192|216 

1 

B.   coli 

1 

0 

0 

0 

0 

0 

+ 

+ 

+ 

, 

. 

+ 

+ 

+ 

2 

» 

1 

i 

0 

0 

0 

4. 

2.2 

__ 

_ 

„_ 













3 

1 

0 

1 

0 

0 

0 

4. 

1.2 

_ 

__ 

~^ 

__ 

— 

— 

— 





4 

}> 

1 

0 

0 

1 

0 

0 

4. 

2 



__ 

«_ 

^  _ 

— 

— 

— 

— 



5 

1 

0 

0 

0 

1 

0 

4. 

2 



_ 

__ 

— 

— 

— 

— 

— 

— 

6 

lt 

1 

0 

0 

0 

0 

1 

— 

0 

4- 

4. 

4. 

_|. 

4_ 

4_ 

4. 

4. 

4. 

7 

P.  vulg.  I 

1 

0 

0 

0 

0 

0 

— 

0 

4- 

4- 

4- 

4- 

4- 

4- 

4. 

4. 

4. 

8 

»          )> 

1 

1 

0 

0 

0 

0 

4. 

1.5 

— 

— 

_ 

— 

— 

— 

— 

— 

— 

9 

»         » 

1 

0 

1 

0 

0 

0 

4_ 

1.5 



— 

— 

— 

— 

— 

— 

— 

— 

10 

>!                )' 

1 

0 

0 

1 

0 

0 

— 

0 

_l_ 

_j_ 

4_ 

-)_ 

4_ 

4_ 

4. 

4. 

4. 

11 

>1                )) 

1 

0 

0 

0 

1 

0 



0 



+ 

_(_ 

4. 

4. 

4. 

4. 

4. 

4. 

12 

»                » 

1 

0 

0 

0 

0 

1 

— 

0 

— 

+ 

4- 

4- 

4- 

4- 

4- 

4- 

4- 

The  results  indicate  that  in  each  case  where  acid  is  produced  by 
the  organism,  there  is  an  inhibition  of  indol  production.    Lactose,  which 


1)  (I)  doubtful. 


—     10    — 

has  no  affect  on  indol  production  by  P.  vulgar  is,  inhibits  its  produc- 
tion in  the  case  of  B.  coli  as  readily  as  does  dextrose.  This  naturally 
leads  to  the  conclusion  that  it  is  the  acid  and  not  the  sugar  which  in- 
hibits the  production  of  indol. 

An  attempt  was  made  to  settle  this  point  more  definitely  ly  inocu- 
lating with  P.  vulgaris  a  sugar  medium  to  which  more  than  enough 
powdered  marble  had  been  added  to  neutralize  the  acid  formed.  It  was 
found,  however,  that  indol  was  not  produced  in  any  case  where  1°/0  or 
more  of  dextrose  had  been  added  to  the  medium.  This  seemed  to  dis- 
prove the  conclusion  that  the  inhibition  of  indol  is  due  to  the  acid 
formed,  but  on  titrating  the  solutions  to  which  the  marble  had  been 
added,  it  was  found  that  the  acid  was  being  produced  much  faster  than 
it  was  neutralized  by  the  marble,  so  that  even  in  this  case  the  acid  was 
probably  the  chief  factor  inhibiting  the  proteolytic  action  of  the  bacteria. 
As  ordinary  marble  may  contain  some  impurities,  it  was  thought  that 
the  impurities  in  this  substance  may  have  had  something  to  do  with 
the  results.  To  avoid  this  source  of  error,  several  flasks  were  prepared, 
each  containing  100  c.  c.  of  a  1%  solution  of  peptone  to  which  were 
added  1%  of  dextrose  and  more  than  enough  chemically  pure  calcium 
carbonate  to  neutralize  all  acid  formed,  together  with  sufficient  of  a  1% 
azolitmin  solution  to  give  a  blue  color.  The  flasks  were  inoculated  with 
Proteus  vulgaris  and  incubated  at  37°.  In  about  24  hours,  the 
litmus  had  turned  red.  A  flask  prepared  in  the  same  manner  was  in- 
oculated with  B.  coli  and  this  also  turned  red.  Flasks  prepared  in  the 
same  way  without  the  addition  of  litmus  and  inoculated  with  B.  coli  or 
P.  vulgaris,  failed  to  produce  indol  in  four  days.  The  calcium  car- 
bonate, while  reducing  the  amount  of  acid  produced,  did  not  keep  the 
solution  alkaline  to  litmus.  Since  acid  sufficient  to  turn  blue  litmus 
red  is  able  to  inhibit  the  action  of  the  tryptic  ferment,  calcium  carbonate 
added  to  a  medium  is  not  sufficient  to  keep  the  degree  of  acidity  low 
enough  for  the  proteolytic  ferment  of  P.  vulgaris  to  act  on  the  peptone. 
To  obviate  this  difficulty  a  flask,  prepared  as  in  the  other  experiments, 
was  inoculated  with  P.  vulgaris  and  the  acid  was  neutralized  from 
time  to  time  by  the  addition  of  a  sterile  solution  of  sodium  carbonate 
to  the  medium.  In  this  flask,  indol  was  formed  as  soon  as  all  the 
sugar  had  been  used  up.  When  lactic  acid  was  added  to  the  peptone 
solution  which  had  been  inoculated  with  P.  vulgaris  so  that  the  so- 
lution was  more  than  0.5  %  acid,  the  production  of  indol  was  inhibited. 
When  less  than  0.5  %  was  added,  however,  the  production  of  indol  was 
not  appreciably  delayed. 

The  above  experiments  seem  to  justify  the  conclusion  that  carbo- 
hydrates are  more  available  to  bacteria  than  are  the  proteins.  In  a  medium 
containing  sugar  and  protein  substances,  the  sugar  is  usually  the  first 
attacked.  When  this  is  used  up  if  the  acid  produced  does  not  retard 
further  action,  the  proteins  are  utilized.  The  inhibition  of  the  proteolytic 
action  is  due  to  the  acid  formed  and  not  to  the  sugar  itself,  for  if  the 
carbohydrate  is  not  fermented  the  proteolytic  action  may  not  be  retarded. 
The  ferment  produced  by  P.  vulgaris  seems  to  be  a  tryptic  ferment. 

Effect  of  carbohydrates  on  gelatin  liquefaction. 

Since  the  addition  of  certain  carbohydrates  to  peptone  solution  in- 
hibited the  production  of  indol  by  P.  vulgaris,  it  was  thought  that 
these  sugars  may  also  inhibit  the  power  of  these  organisms  to  liquefy 


11    — 


gelatin.  Gelatin  was  made  up  in  bulk  and  distributed  to  a  series  of 
flasks.  One  per  cent,  of  carbohydrate  was  added  to  each  flask,  and  the 
gelatin  in  the  flasks  was  poured  into  tubes  of  equal  diameter,  so  that 
each  tube  contained  gelatin  to  a  depth  of  50  m.  m.  After  sterili- 
zation, each  tube  was  inoculated  with  the  organism  to  be  tested  by 
spreading  a  suspension  of  the  organism  over  the  surface  of  the  gelatin. 
The  tubes  were  then  placed  in  the  incubator,  kept  at  22°  C  and  allowed 
to  remain  for  thirty  days.  At  the  end  of  the  incubation  period,  the 
amount  of  liquefaction  was  determined  by  measuring  the  depth  of  the 
gelatin  which  had  been  reduced  to  a  liquid  state. 

The  results  of  the  experiments  are  given  in  the  table  which  follows. 

Table  No.  5. 

Table  showing    the  amount  of  liquefaction   in   millimeters   in    carbo- 
hydrate gelatin  by  members  of  the  Proteus  group. 


No. 

Plain 
Gel. 

Dex- 
trose 

Saccha- 
rose 

Levu- 
lose 

Man- 
nose 

Galac- 

tose 

Mal- 
tose 

Lac- 
tose 

Eaffi- 
nose 

Man- 
nite 

1 

Prot.  vulg.  I 

50 

5 

_ 

_ 

50 

1 

40 

2 

*      II 

50 

— 

— 

3 

— 

— 

— 

50 

— 

40 

3 

„      mirabilis 

23 

3 

— 

— 

— 

— 

— 

20 

— 

50 

4 

„      Zenkeri 

— 

— 

— 

— 

— 

— 

— 

— 

— 

— 

5 

B.  cloacae  I 

5 

8 

18 

25 

4 

50 

11 

10 

5 

8 

6 

„       11 

11 

35 

50 

8 

4 

50 

30 

10 

16 

50 

7 

,,       HI 

11 

12 

35 

25 

25 

40 

30 

30 

25 

50 

8 

„     iv 

10 

20 

20 

9 

1 

50 

10 

10 

15 

50 

9 

P.  vulgaris 

30 

2 

2 

19 

3 

5 

3 

50 

20 

50 

10 

P.  mirabilis 

— 

— 

— 

— 

— 

— 

— 

— 

— 

— 

11 

P.  Zenkeri 

— 

— 

— 

— 

— 

— 

— 

— 

— 

— 

12 

B.  cloacae 

12 

12 

19 

10 

15 

40 

14 

20 

4 

50 

13 

B.  vulgaris 

19 

— 

— 

— 

— 

— 

— 

50 

— 

50 

14 

B. 

19 

— 

— 

— 

— 

~-^- 

— 

15 

— 

50 

15 

B.  Zopfi 

•  — 

— 

— 

~ 

— 

— 

— 

— 

— 

— 

16 

B.  vulgaris 

21 

— 

— 

2 

— 

— 

— 

35 

— 

50 

17 

B.  mirabilis 

30 

— 

— 

4 

2 

5 

3 

50 

— 

50 

18 

B.  Zenkeri 

— 

— 

— 



— 

— 

— 

— 

— 

— 

19 

B.  cloacae 

25 

5 

12 

10 

15 

40 

16 

25 

17 

40 

20 

P.  vulgaris 

38 

— 

— 

— 

1 

3 

— 

50 

— 

50 

21 

P.  mirabilis 

— 

— 

— 

— 



— 

— 

— 

— 

— 

22 

P.  Zenkeri 







— 

— 

— 



.... 

— 

— 

23 

P.  Zopfi 

— 

— 

— 

— 

— 

— 

— 

— 

— 

— 

24 

B.  cloacae 

20 

26 

50 

50 

50         50 

25 

18 

25 

50 

The  table  shows  that  dextrose,  saccharose,  levulose,  mannose,  galac- 
tose,  maltose ,  and  raffinose,  tend  to  inhibit  the  .liquefaction  of  gelatin 
by  all  strains  of  P.  vulgaris  and  by  P.  mirabilis,  3  and  17,  while 
gelatin  containing  lactose  or  mannite  do  not  retard  the  liquefaction  of 
gelatin.  All  of  the  strains  of  P.  Zenkeri,  P.  Zopfi  i,  and  mirabilis, 
with  the  exception  of  P.  mirabilis  3  and  17,  did  not  liquefy  gelatin 
in  any  of  the  tubes,  even  when  allowed  to  remain  in  the  incubator  for 
three  months.  It  was  found  that  sugars  which  inhibited  the  gelatin 
liquefaction  by  Proteus  vulgaris  were  fermented  by  that  organism. 

To  find  out  whether  acid  was  produced  by  the  fermentation  of  the 
sugars  in  the  gelatin  tubes,  a  second  series  of  sugar  gelatin  tubes  were 
prepared  and  a  few  drops  of  1°/0  solution  of  azolitmin  were  added  to 
each,  as  well  as  to  the  nutrient  gelatin  control.  The  tubes  were  inocu- 
lated with  P.  vulgaris  as  in  the  previous  experiment  and  incubated 
at  22  °  C.  In  every  tube  which  showed  liquefaction,  the  litmus  remained 


1  9 

j.^ 

blue,  but  in  all  cases  where  gelatin  was  not  liquified,  turned  decidedly 
red.  The  acid  formed  by  the  fermentation  of  the  sugar  is  probably 
therefore,  the  chief  factor  at  work  in  preventing  gelatin  liquefaction  by 
P.  vulgaris. 

In  view  of  the  experiments  of  Ogata  (29),  Labor de  (30),  Gold- 
thwait  (31)  and  Lindsay  (32),  in  which  these  authors  seem  to  have 
demonstrated  that  the  addition  of  carbohydrates  to  artificial  digestion 
solutions  retards  the  digestion  of  protein  substances,  it  would  seem  that 
sugars  themselves  should  retard  the  proteolytic  action  of  bacterial  fer- 
ments as  well.  Bearing  in  mind  that  these  authors,  experiments  were 
conducted  with  peptic  ferments,  the  idea  might  be  entertained  that 
sugars  likewise  exert  some  retarding  effect  in  the  presence  of  tryptic 
ferments  such  as  are  produced  by  P.  vulgaris.  That  the  mere  presence 
of  sugar  is  not  effective,  however,  is  conclusively  demonstrated  by  the 
following  experiments. 

A  flask  containing  50  c.  c.  of  nutrient  gelatin  with  a  reaction  neutral 
to  phenolphthalein  was  inoculated  with  P.  vulgaris  I  and  allowed  to 
stand  until  completely  liquefied.  A  portion  of  the  liquefied  gelatin  was 
passed  through  a  hard  filter  to  remove  the  bacteria.  Two  series  of 
flasks  were  prepared  as  indicated  in  the  following  table.  To  each  of  the 
first  series  of  flasks  one  c.  c.  of  the  unfiltered  gelatin  was  added,  while 
to  each  of  the  second  series  of  flasks  1  c.  c.  of  the  filtered  gelatin  was 
added.  Both  series  were  allowed  to  stand  under  the  same  conditions. 
The  results  for  the  sake  of  convenience  are  given  in  tabulated  form. 

1.  50  c.  c.  nutrient  gelatin  +  2  °/0  dextrose  +1  c.  c.  filtered  gelatin  =  no  liquefaction  in 

20  days. 

2.  50  c.  c.         „  „       +  2  %        TJ         +1  c- c-  filtered  gelatin  =  liquefaction  in  4 

days. 

3.  50  c.  c.         „  „       +  2  %        »         +  1  c.  c.  unfiltered  gelatin  +  1  c.  c.    filtered 

gelatin  =  no  liquefaction  in  8  days. 

4.  50  c.  c.         „  „       +2  %  lactose     +1  c.  c.  unfiltered  gelatin  —  liquefaction  in 

4  days. 

5.  50  c.  c.         „  „       4-  2  °/0       n          +1  c.  c.  filtered  gelatin  =  liquefaction  in  4 


6.  50  c.  c.         „  „  +1  c.  c.  unfiltered  gelatin  =  liquefaction   in 

4  days. 

7.  50  c.  c.         „  „  +  1  c.  c.  filtered  gelatin  =  liquefaction  in  4 

days. 

8.  50  c.  c.         „  „       +2  %  HC1         +1  c.  c.  filtered  gelatin  =  no  liquefaction  in 

10  days. 

All  the  flasks  were  tested  at  the  completion  of  the  experiments  and 
pure  cultures  of  P.  vulgaris  were  found  in  all  of  the  flasks  to  which 
the  unfiltered  gelatin  had  been  added  while  those  flasks  which  had  re- 
ceived the  filtered  gelatin  proved  sterile.  Flasks  one  and  three  deve- 
loped an  acid  reaction  while  the  reaction  of  the  other  flasks  remained 
unchanged. 

The  results  are  striking  and  conclusive.  In  those  flasks  to  which 
acid  had  been  added  or  which  developed  an  acid  reaction  no  liquefaction 
resulted,  while  in  the  flasks  in  which  no  acid  was  produced  liquefaction 
took  place  at  a  fairly  uniform  rate.  There  can  be  but  one  interpretation 
of  these  results:  the  liquefaction  of  the  gelatin  was  inhibited  by  the 
acid.  We  know  from  the  above  experiments  that  dextrose  has  no  effect 
upon  the  proteolytic  ferment  when  formed.  If  to  the  dextrose  gelatin 
we  add  in  addition  to  the  filtered  gelatin  a  suspension  of  Proteus  vul- 
garis we  get  no  liquefaction.  In  this  case,  however,  the  bacteria  attacked 


—     13    — 

the  dextrose,  acid  was  formed  and  this  acid  inhibited  the  proteolytic 
action.  Whether  or  not  the  presence  of  dextrose  or  the  acid  produced 
from  dextrose  prevents  the  formation  of  the  proteolytic  enzyme  is  not 
evident  from  these  experiments,  though  it  seems  probable  that  dextrose 
alone  would  not  be  capable  of  exerting  such  an  influence. 

B.  cloacae,  unlike  P.  vulgar  is,  is  able  to  liquefy  gelatin  in  the 
presence  of  all  the  sugars  tried.  It  was  noted,  however,  that  the  lique- 
faction in  the  case  of  B.  cloacae  did  not  begin  as  quickly  in  the 
sugar  gelatin  tubes  as  in  the  nutrient  gelatin  tubes,  though  when  it  did 
begin  it  proceeded  further  in  some  cases.  An  initial  acid  production 
takes  place  in  sugar  gelatin  tubes  inoculated  with  B.  cloacae  since 
litmus  added  to  these  tubes  turned  red  after  48  hours  incubation  at 
22°  C.  Evidently,  a  small  amount  of  acid  does  not  interfere  with  the 
proteolytic  action  of  B.  cloacae  to  any  great  extent.  Since  it  was 
shown  in  a  previous  paper  (33)  that  the  initial  acidity  produced  by 
B.  cloacae  in  a  sugar  protein  medium  may  be  followed  by  alkalinity, 
it  is  possible  that  the  amido  compounds  produced  by  the  proteolytic 
action  of  this  organism  tend  to  neutralize  the  acid  produced,  and  thus 
keep  the  percentage  of  acid  low  enough  to  enable  the  proteolytic  ferment 
to  work,  or  the  proteolytic  ferment  of  B.  cloacae  may  be  more  resistant 
to  acid  than  is  the  ferment  of  P.  vulgar  is.  This  is  suggestive  that 
the  proteolytic  ferment  of  B.  cloacae  is  more  peptic  than  tryptic. 

An  attempt  was  made  to  cause  the  non-gelatin  liquefiers  to  liquefy 
gelatin  by  growing  them  in  nutrient  gelatin  for  varying  lengths  of  time 
under  different  degrees  of  temperature.  Some  gelatin  cultures  were 
placed  in  the  37°  incubator  and  allowed  to  remain  there  for  six  days. 
The  gelatin  was  then  placed  in  the  ice  box  to  see  if  it  would  solidify 
again.  The  tubes  on  solidifying,  were  plated  out  in  gelatin  and  ten 
characteristic  colonies  were  selected  and  inoculated  into  gelatin  tubes. 
These  were  allowed  to  stand  for  six  days  and  again  plated  out  and 
selections  made  from  the  colonies  on  the  plate.  Although  this  process 
was  carried  on  through  several  transfers,  not  a  single  culture  was 
obtained  which  would  liquefy  nutrient  gelatin  when  allowed  to  stand 
at  22°  for  30  days.  All  the  gelatin  tubes  inoculated  with  the  non- 
liquefiers  and  kept  in  the  37°  incubator  solidified  again  when  placed  in 
the  ice  box  for  a  short  time.  The  colonies  on  the  gelatin  plates  were 
characteristic  only  when  the  gelatin  was  moist.  If  the  percentage  of 
gelatin  in  the  medium  was  high,  the  characteristic  Proteus  colonies 
did  not  always  develop. 

The  fact  that  Proteus  Zenkeri  and  Proteus  mirabilis  do 
not  liquefy  gelatin  readily  or  at  all  is  hardly  sufficient  reason  to  classify 
these  organisms  as  species  distinct  from  Proteus  vulgaris.  Some 
authors  have  made  the  observation  that  a  species  of  P.  Zenkeri  which 
had  repeatedly  failed  to  liquefy  gelatin  suddenly  developed  this  power, 
while  Smith  (5)  was  able  to  obtain  a  non-gelatin  liquefier  from  P. 
vulgaris.  These  facts  would  indicate  that  P.  Zenkeri  and  P.  mira- 
bilis and  Zopfii  are  varieties  of  P.  vulgaris  which  have  lost  many 
of  their  enzymotic  powers  but  have  developed  no  new  characteristics 
which  would  be  sufficient  to  call  them  distinct  species.  P.  Zopfii  and 
P.  Zenkeri,  together  with  the  strains  of  P.  mirabilis  which  do  not 
liquefy  gelatin,  all  belong  undoubtedly  to  the  same  variety,  P.  Zenkeri, 
and  the  names  Zopfii  and  mirabilis  could  very  well  be-  done  away 
with.  The  strains  of  P.  mirabilis  are  at  best  intermediate  forms 


—     14    — 

between  vulgaris  and  Zenkeri,  which  need  but  cultivation  to  turn 
them  into  P.  vulgaris  on  the  one  hand  or  P.  Zenkeri  on  the  other. 
The  morphological  characteristics  are  of  little  use  in  the  differentiation 
of  these  organisms  since  under  artificial  conditions  either  may  produce 
short  rods  or  longs  filaments,  according  to  the  kind  of  media  used  and 
the  condition  under  which  they  are  grown.  P.  vulgaris  in  all  strains 
was  negative  to  Gram,  while  P.  Zenkeri  was  usually  positive,  but 
even  this  characteristic  is  so  variable  that  it  has  no  characteristic  value. 
None  of  the  species  of  Proteus  examined  produced  any  pigment. 
This  test  was  made  on  potato,  Heinemanns  synthetic  medium,  and 
on  agar,  to  which  1  %  of  tyrosin  had  been  added.  In  the  latter  medium, 
P.  vulgaris  produced  a  dark  coloration  in  the  medium  in  about  24  hours, 
but  no  pigment  was  developed. 

Conclusions. 

1.  Selection   of  slight  variations  in  acid  production  in  sugar  broth 
by  P.  vulgaris  tailed  to  produce  any  variation  in  the  acid  producing 
properties  of  this  organism. 

2.  P.  vulgaris,  though  not  able  to  ferment  lactose  under  aerobic 
conditions,  does  so  when  cultivated  under  anaerobic  conditions. 

3.  Carbohydrates,   when    fermented   by  P.   vulgaris   or   B.  coli, 
inhibit  the  production  of  indol,  by  these  organisms.    The  inhibition  is, 
however,  due  to  the  acid  formed  and  not  to  the  carbohydrate  itself. 

4.  The  addition  of  more  than  0,5  %  °f  lactic  acid  to  the  culture  medium 
inhibits  the  production  of  indol. 

5.  The  acid  formed  by  the  fermentation  of  carbohydrates  inhibits  to 
a  certain  degree  the  liquefaction  of  gelatin  by  members  of  the  Proteus 
group. 

6.  The   ferment  formed   by  P.  vulgaris   seems   to  be  a  tryptic 
ferment,  while  that  of  B.  cloacae  is  suggestive  of  a  peptic  ferment. 

7.  P.  Zopfii  and  Zenkeri  are  probably  one  and  the  same  variety, 
and  P.  mirabilis  seems  to  be  an  intermediate  form  between  Zenkeri 
and  vulgaris  which  differs  in  the  intensity  of  its  biochemical  reactions, 
but  not  in  the  quality.    Some  strains  of  mirabilis  examined  gave  similar 
biochemical  reactions  to  those  of  P.  Zenkeri,   while  others  resembled 
P.  vulgaris  very  closely. 

The  writer  takes  great  pleasure  in  thanking  Professors  E.  0.  Jordan 
and  Norman  MacL.  Harris  for  their  suggestions  and  encouragement 
in  this  work. 

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—     15    — 

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